CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

The frequency of double-positive thymocytes expressing an alphabeta TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing alphabeta T-cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T-cell compartment size. Scid recipients were inoculated with various ratios of TCR Calpha(0/0) and wild-type bone marrow (BM) stem cells. Increasing the frequency of TCR Calpha(0/0) thymocytes at steady-state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12-14 weeks following BM inoculation, the frequency of TCR Calpha(0/0) DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild-type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency-dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady-state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady-state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.     

GATA-3 expression is controlled by TCR signals and regulates CD4/CD8 differentiation

GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

A cortisone sensitive CD3low subset of CD4+CD8- thymocytes represents an intermediate stage in intrathymic repertoire selection.

Two populations of CD4 single positive (SP) thymocytes were found in transgenic mice bearing class I-restricted Mls-1a reactive (V beta 8.1) TCR genes in the absence of the restriction element. CD3high CD4 SP cells were deleted in the presence of Mls-1a and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted in the presence of Mls-1a and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data indicate that CD3low CD4 SP thymocytes represent an intermediate stage in the transition from CD3low double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may hve undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotype.     

TCR and Notch signaling in CD4 and CD8 T-cell development

Summary:  The generation of CD4 and CD8 αβ T-cell lineages from CD4⁺CD8⁺ double-positive (DP) thymocyte precursors is a complex process initiated by engagement of major histocompatibility complex (MHC) by T-cell receptor (TCR) and coreceptor. Quantitative differences in TCR signaling induced by this interaction impose an instructional bias on CD4/CD8 lineage commitment that must be reinforced by MHC recognition and TCR signaling over subsequent selection steps in order for the thymocyte to progress and mature in the adopted lineage. Our studies show that the transmembrane receptor Notch plays a role in this process by modifying TCR signal transduction in DP thymocytes. In this review, we consider the functional relationship of TCR and Notch signaling pathways in the selection and specification of CD4 and CD8 T-cell lineages.     

In vitro differentiation and commitment of CD4+ thymocytes to the CD4+ lineage without TCR engagement

Thymocyte positive selection is based on protection of immatureCD4/CD8 double-positive (DP) thymocytes from apoptosis and theirdifferentiation into CD4 or CD8 single-positive (SP) cells.Intracellular signals essential for positive selection appearto be induced through the TCR and some of the accessory moleculesincluding LFA-1, CD4 and CD8 upon Interaction with thymic stromalcells. The signals, however, still remain to be identified.Since physiological levels of glucocorticoids potentially induceor enhance thymocyte apoptosis even in vivo, the signals arelikely to inhibit the apoptotic effect of glucocorticoids. Wehave previously shown that proper cross-linking of TCR-CD3 withLFA-1, CD4 or CD8 inhibited glucocortlcold-lnduced thymocyteapoptosis in vitro, and that a proper combination of the calciumionophore, ionomycin and the protein kinase C (PKC) activator,phorbol 12-myrlstate 13-acetate (PMA), mimicked the inhibitoryeffect. Here we determined whether this combination of ionomycinand PMA induces differentiation of isolated DP thymocytes fromnormal and TCR transgenic mice. We found that pretreatment ofDP thymocytes with ionomycin and PMA followed by 1 day cultureof the cells without the reagents resulted in the differentiationof the cells into CD4 SP and CD4⁺ CD8^lo T cells that have mostlycommitted to the CD4 lineage. The changes in expression of otherdifferentiation markers were also in good accordance with thoseassociated with positive selection, except the final maturation.The results indicate that moderate and transient increases inintracellular Ca²⁺ level and PKC activity induce differentiationand commitment of DP thymocytes to the CD4 lineage, and suggestedthat the biochemical pathway leading to positive selection isbased on a similar mechanism.     

Alterations during positive selection in the thymus of nackt CD4-deficient mice

The T-cell repertoire is shaped by the positive and negative selection of immature CD4(+) CD8(+) double positive (DP) thymocytes. Positive selection of DP T cells to the CD4(+) CD8(-) and CD4(-) CD8(+) simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4(+) CD8(-) T-cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69(+) and CD5(+) cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4(+) SP cells.     

Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes

To understand how CD8 expression is regulated during the transition process from CD4+8+ (CD4 and CD8 double positive, DP) to CD4-8+ (CD8 single positive, CD8SP) cells in the thymus, the involvement of Runx proteins in the alteration of chromatin configuration was investigated. Using the chromatin immunoprecipitation assay, we first demonstrated that Runx proteins bind to the stage-specific CD8 enhancer, as well as the CD4 silencer, in CD8SP thymocytes. Among Runx family members, Runx3 expression was initiated in DP thymocytes receiving a positive selection signal and increased in concert with differentiation to the CD8SP stage. Furthermore, reactivation of the CD8 gene, as well as CD4 silencing, was suppressed in positively selected thymocytes of Runx dominant-negative transgenic mice. These results suggest that Runx proteins, especially Runx3, are involved in lineage specification of CD8 T cells and provide important information for understanding the mechanism for the mutually exclusive expression of coreceptors in mature thymocytes.     

Maturation of CD4-8- alpha/beta TCR+ T cells induced by CD2-stimulation in vivo and in vitro.

Newly generated ('virgin') rat thymocytes of the immature CD4+8+ double positive (DP) subset were treated in suspension culture for 2 days with the stimulatory pair of anti-CD2 monoclonal antibodies OX-54 and OX-55. Approximately 50% of the recovered cells had downregulated CD4 and CD8 and upregulated the T cell antigen receptor (TCR). CD2-stimulated, but not control thymocytes proliferated in response to TCR plus IL-2 stimulation. In vivo, postnatal injection of OX-54/55 led to a dramatic and selective increase in functionally mature CD4-CD8- double negative (DN) alpha/beta--TCR(high) thymocytes and peripheral T cells. These findings show that CD2 stimulation can promote T cell differentiation and suggest that DN TCR(high) thymocytes can be generated from DP thymocytes via alternative pathways of T cell maturation.     

V?Gene Family Usage in Spontaneous Lymphomas of AKR Mice: Evidence for Defective Clonal Deletion

T-cell receptor (TCR) ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4⁺8⁺) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V ß gene family usage, 2 out of 14 lymphomas expressed V ß6. Normally, V ß6⁺ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V ß6⁺ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.     

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes

T-cell differentiation in the thymus depends on positive selection of CD4+CD8+ double positive (DP) thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows maturation of only those thymocytes that are capable of self-peptide-MHC recognition. Thymocytes that fail to bind self-peptide-MHC die by apoptosis. An important question in thymocyte differentiation is whether co-stimulation is required for positive selection and on which cells co-stimulatory molecules may be expressed in the thymus. The vascular cell adhesion molecule (VCAM-1) and the intercellular cell adhesion molecule (ICAM-1) are known to be potent co-stimulatory molecules in activation of peripheral T-cells by interacting with the integrins VLA-4 and LFA-1, respectively. We were prompted to investigate whether VCAM-1 and ICAM-1 may also act as co-stimulators during selection of thymocytes. By using recombinant proteins of murine VCAM-1 and ICAM-1 fused to the Fc region of human IgG1 (rVCAM-1, rICAM-1) we examined the capacity of VCAM-1 and ICAM-1 to act as co-stimulatory molecules in positive selection in vitro. Triggering the CD3/TCR complex together with co-stimulation applied by rVCAM-1 or rICAM-1 induced the generation of CD4+ single positive (SP) thymocytes from CD4+CD8+ DP thymocytes whereas either signal alone did not result in generation of CD4+ SP thymocytes. VCAM-1 and ICAM-1 act therefore as co-stimulatory molecules in thymocyte positive selection in vitro. The generation of CD4+ SP cells is accompanied by cell survival both when it was co-stimulated with rVCAM-1 and with rICAM-1. Importantly we show here that VCAM-1 expression in the murine thymus is restricted to cortical F4/80 positive hematopoietic antigen presenting cells (hAPC) present exclusively in the cortex whereas expression of ICAM-1 has been reported on the epithelium both in cortex and medulla. This suggests that not only the cortical epithelium may use the co-stimulatory molecule ICAM-1 to mediate positive selection, but also cortical hAPCs may contribute to positive selection of thymocytes by using the co-stimulator VCAM-1.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

Glucocorticoid (GC) sensitivity and GC receptor expression differ in thymocyte subpopulations

Positive and negative selection steps in the thymus prevent non-functional or harmful T cells from reaching the periphery. To examine the role of glucocorticoid (GC) hormone and its intracellular receptor (GCR) in thymocyte development we measured the GCR expression in different thymocyte subpopulations of BALB/c mice with or without previous dexamethasone (DX), anti-CD3 mAb, RU-486 and RU-43044 treatment. Four-color labeling of thymocytes allowed detection of surface CD4/CD8/CD69 expression in parallel with intracellular GCR molecules by flow cytometry. Double-positive (DP) CD4+CD8+ thymocytes showed the lowest GCR expression compared to double-negative (DN) CD4-CD8- thymocytes and mature single-positive (SP) cells. DX treatment caused a concentration-dependent depletion of the DP cell population and increased appearance of mature SP cells with reduced GCR levels. GCR antagonists (RU-486 or RU-43044) did not influence the effect of DX on thymocyte composition; however, RU-43044 inhibited the high-dose GC-induced GCR down-regulation in SP and DN cells. GCR antagonists alone did not influence the maturation of thymocytes and receptor numbers. Combined low-dose anti-CD3 mAb and DX treatment caused an enhanced maturation (positive selection) of thymocytes followed by the elevation of CD69+ DP cells. The sensitivity of DP thymocytes with a GCRlow phenotype to GC action and the ineffectiveness of the GCR antagonist treatment may reflect a non-genomic GC action in the thymic selection steps.     

CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.